ABSTRACT
BACKGROUND: The negative effect of tumor necrosis factor-alpha (TNF-alpha) on heart contraction, which is mediated by sphingosine, is a major component in heart failure. Because the cellular level of glutathione may limit sphingosine production via the inhibition of the Mg-dependent neutral sphingomyelinase (N-SMase), we hypothesized that cardiac glutathione status might determine the negative contractile response to TNF-alpha. METHODS AND RESULTS: We examined the effects of TNF-alpha in isolated cardiomyocytes obtained from control rats or rats that were given the glutathione precursor N-acetylcysteine (NAC, 100 mg IP per animal). In cardiomyocytes obtained from control rats, 25 ng/mL TNF-alpha increased reactive oxygen species generation and N-SMase activity (500% and 34% over basal, respectively) and decreased the amplitude of [Ca(2+)](i) in response to electrical stimulation (22% below basal). NAC treatment increased cardiac glutathione content by 42%. In cardiomyocytes obtained from NAC-treated rats, 25 ng/mL TNF-alpha had no effect on reactive oxygen species production or N-SMase activity but increased the amplitude of [Ca(2+)](i) transients and contraction in response to electrical stimulation by 40% to 50% over basal after 20 minutes. This was associated with a hastened relaxation (20% reduction in t(1/2) compared with basal) and an increased phosphorylation of both Ser(16)- and Thr(17)-phospholamban residues (260% and 115% of maximal isoproterenol effect, respectively). CONCLUSIONS: It is concluded that cardiac glutathione status, by controlling N-SMase activation, determines the severity of the adverse effects of TNF-alpha on heart contraction. Glutathione supplementation may therefore provide therapeutic benefits for vulnerable hearts.
Subject(s)
Acetylcysteine/pharmacology , Calcium/metabolism , Glutathione/analogs & derivatives , Myocytes, Cardiac/physiology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Calcium-Binding Proteins/metabolism , Cells, Cultured , Glutathione/metabolism , Glutathione/pharmacology , Male , Myocardial Contraction , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Sphingomyelin Phosphodiesterase/metabolism , Tumor Necrosis Factor-alpha/toxicityABSTRACT
We have recently established that enhancement of intracellular calcium cycling and contraction in response to beta2-adrenergic receptor (beta2-AR) stimulation exclusively relies on the activation of the cytosolic phospholipase A2 (cPLA2) and arachidonic acid production, via a pertussis toxin-sensitive G protein (possibly Gi), in embryonic chick cardiomyocytes. We aimed to investigate the relevance of the beta2-AR/Gi/cPLA2 pathway in the human myocardium. In left ventricular biopsies obtained from explanted hearts, beta2-AR stimulation exerted either an inhibition of cPLA2 that was insensitive to pertussis toxin (PTX) treatment, or an activation of cPLA2, sensitive to PTX treatment. In right atrial appendages from patients who were undergoing open heart surgery, we demonstrated that beta2-AR-induced activation of cPLA2 was favored in situations of altered beta1-AR and/or beta2-AR/adenylyl cyclase (AC) stimulations. Alterations were characterized by an increase in EC50value of norepinephrine and a decrease in the maximal AC activation in response to zinterol, respectively. Quantitative reverse transcription-polymerase chain reaction analyses highlighted a positive correlation between the expression of AC5 and AC6 mRNAs in human cardiac atria, which suggested that functional alterations in AC responses were unlikely to be related to changes in the AC5/AC6 mRNA ratio. In addition, the shift from the cyclic AMP to the arachidonic acid pathway was not supported at the transcriptional level by opposite regulation of AC and cPLA2mRNAs expression. This study gives the first evidence of the recruitment of cPLA2by beta2-ARs in the human heart and suggests that the Gi/cPLA2pathway could substitute for a deficient Gs/AC pathway in mediating beta2-AR responses.
Subject(s)
Adenylyl Cyclases/metabolism , Arachidonic Acid/metabolism , Cyclic AMP/metabolism , Heart/physiology , Isoenzymes/metabolism , Receptors, Adrenergic, beta-2/physiology , Adenylyl Cyclases/genetics , Atrial Appendage , Group IV Phospholipases A2 , Heart Ventricles/enzymology , Humans , Isoenzymes/genetics , Phospholipases A/metabolism , Phospholipases A2 , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Statistics as TopicABSTRACT
Tumor necrosis factor (TNF)-alpha has a biphasic effect on heart contractility and stimulates phospholipase A2 (PLA2) in cardiomyocytes. Because arachidonic acid (AA) exerts a dual effect on intracellular Ca2+ concentration ([Ca2+]i) transients, we investigated the possible role of AA as a mediator of TNF-alpha on [Ca2+]i transients and contraction with electrically stimulated adult rat cardiac myocytes. At a low concentration (10 ng/ml) TNF-alpha produced a 40% increase in the amplitude of both [Ca2+]i transients and contraction within 40 min. At a high concentration (50 ng/ml) TNF-alpha evoked a biphasic effect comprising an initial positive effect peaking at 5 min, followed by a sustained negative effect leading to 50-40% decreases in [Ca2+]i transients and contraction after 30 min. Both the positive and negative effects of TNF-alpha were reproduced by AA and blocked by arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2. Lipoxygenase and cyclooxygenase inhibitors reproduced the high-dose effects of TNF-alpha and AA. The negative effects of TNF-alpha and AA were also reproduced by sphingosine and were abrogated by the ceramidase inhibitor n-oleoylethanolamine. These results point out the key role of the cytosolic PLA2/AA pathway in mediating the contractile effects of TNF-alpha.
